大肠杆菌胞嘧啶脱氨酶基因的克隆及其真核表达载体的构建

The cloning of cytosine deaminase gene of Escherichia coli and the construction of eukaryotic expression vector

目的 克隆大肠杆菌胞嘧啶脱氨酶 ( CD)基因 ,构建真核表达载体 ,本研究拟探索该基因在肿瘤基因治疗中的应用基础。方法 根据 Gen Bank数据库提供的 CD基因核苷酸序列 ,设计并合成一对引物 ,采用 PCR方法 ,从大肠杆菌基因组 DNA中扩增出 CD基因 ,并与 pc DNA3.1定向连接 ,构建受控于人巨细胞病毒启动子的重组真核载体 pc DNA3.1- CD,并用限制性内切酶、PCR和 DNA测序进行鉴定。结果 克隆了大肠杆菌 CD基因 ,并构建了真核表达载体 ,经限制性内切酶酶切、PCR扩增和 DNA测序证实了其正确性。结论 pc DNA 3.1- CD真核表达载体构建成功。

Objective To clone cytosine deaminase(CD) gene of Escherichia coli and to construct its eukaryotic expression vector Methods A pair of primers were designed and synthesized based on CD gene sequence in GenBank And CD gene was cloned from Escherichia coli genome by PCR amplification The PCR products were cloned into a eukaryotic plasmid pcDNA3 1 to construct the recombination expression vector which was controlled by CMV promoter The recombinant plasmid was analyzed and identified by PCR, restriction digest and sequencing Results The CD gene was cloned and eukarytoic expression vector (pcDNA3 1 CD) was constructed It was demonstrated that CD gene was properly inserted into the vector and the sequence was confirmed with restriction digest, PCR amplification and sequencing Conclusion The eukaryotic expression vector with CD gene (pcDNA3 1 CD) was successfully constructed